Loading...
Thesis
Browse
7 results
Search Results
ThesisItem Restricted Genome wide selection for rapid introgression of productivity traits from Oryza rufipogon into O. sativa(Punjab Agricultural University, Ludhiana, 2019) Malik, Palvi; Kumari NeelamThe genome wide selection for rapid introgression of productivity traits was carried out on a diverse set of 346 accessions of Oryza rufipogon. Population structure investigated using 2271 SNPs in O. rufipogon, indicated a weakly differentiated population which further classified into six sub-populations. Linkage disequilibrium decayed to its half-maximum at 10 kb for Oryza rufipogon and 60 kb for Oryza sativa. Genome wide association analysis was carried out using a set of 44,109 SNPs for seven traits, i.e, plant height, culm thickness, panicle length, number of primary branches, grain length, grain width, hundred grain weight. The study revealed 37 strong SNP associations, out of which 13 SNPs were associated with multiple traits. A total of 14 SNPs localized in 12 previously reported QTL regions of the concerned traits. Genomic selection was carried out on 1751 backcross lines derived from 11 different backcross families. Bayes B and Bayes C were used to build models in training population, comprised of 1223 and 1215 lines for panicle length and plant height, respectively. GS accuracy for above mentioned traits came out to be 0.51 and 0.72, respectively. In conclusion, the study revealed very subtle population structure with low levels of differentiation in Oryza rufipogon population. The analysis yielded 23 novel SNPs, which might serve as putative candidates for further investigation of agronomically important traits. The backcross progenies with good genomic estimated breeding values can be further utilized in breeding programs for improving yield related traits.ThesisItem Open Access Fine mapping of qtl-qslb.pau-3.04 for southern leaf blight resistance in maize (Zea mays L.)(Punjab Agricultural University, Ludhiana, 2019) Kirandeep Kaur; Vikal, YogeshSouthern leaf blight (SLB) caused by Cochliobolus heterostrophus (Drechsler), is a serious disease throughout the world where maize is grown under warm and humid conditions, leading to enormous yield losses. Exploiting genes and quantitative trait loci (QTLs) related to SLB is helpful for improving fungal resistance. In earlier study at Punjab Agricultural University, Ludhiana a set of 325 F2:3 families and F4 progenies derived from the cross of CM139 as the resistant (female) parent and CM140 as the susceptible (male) parent were phenotyped for resistance to SLB under field conditions. A total of 172 polymorphic SSR markers were genotyped on F2 population.Three probable QTL viz. qSLB2.1, qSLB3.1, qSLB3.2 were detected for SLB resistance in bins of 2.05-2.08, 3.04 and 3.06-3.09. The marker interval phi099-umc1729 spanning qSLB3.1 was considered as major putative QTL. In present study, the QTL qSLB3.1 spanning region was saturated with more SSR markers on 298 RIL population of the same cross. The detected QTL region (13.7 cM) was fine mapped to 1.2 cM region flanked with marker MSSR1-MSSR20 explaining phenotypic variance of 15.1 per cent at log-likelihood of 6.9. The 1.2 cM region corresponds to 269.3 Kb and comprises only six candidate genes. The candidate gene based markers were designed, to study the differential expression patterns of candidate genes through quantitative real-time PCR (qRT-PCR). Two of the candidate genes viz. GRMZM2GO88371 and GRMZM5G862219 showed 3.48 and 6.40 fold higher expression in CM140 at 48h and 72h respectively. GRMZM2GO88371 and GRMZM5G862219 have their biological function in lipid metabolism and β oxidation respectively that is involved in the defense pathway and may be one of the potential candidate genes conferring resistance against C. heterostrophus. The SLB QTL linked flanking markers were employed for mobilization of qSLB3.1 QTL into the background of CM140 through marker assisted backcross breeding (MABB). The F1s of cross (CM139 X CM140) were backcrossed to recurrent parent to generate BC1F1 population. A total of 64 plants out of 420 were selected on the basis of foreground selection. Recombinant selection for the carrier chromosome was done to identify single and double recombinants. The plants having maximum recurrent parent recovery for carrier chromosome were selected and backcrossed with CM140 to generate BC2F1 generation. The data generated from this study can serve as valuable genomic resource for maize breeding programmes. It will enable the researcher to multi-thronged and focused approaches for sustainable development of new genotypes by pyramiding it with other desirable genes using MABB.ThesisItem Open Access Heterologous expression and characterization of antifungal chitinase and glucanase genes from Trichoderma viride in Escherichia coli(Punjab Agricultural University, Ludhiana, 2019) Ramanjeet Kaur; Sandhu, Jagdeep SinghThe endochitinase and β-1,3-glucanase genes isolated from Trichoderma spp. were expressed in Escherichia coli strains Rosetta-gami 2 (DE3) and BL21 (DE3) pLysS using His-tagged pET28a+ and pET6xHN-C vectors. The expression of endochitinase and β-1,3-glucanase recombinant proteins was optimized by varying the incubation conditions, such as incubation temperature, incubation time and inducer concentration. The maximum expression (86.23 ng/ml) of endochitinase was observed with 0.4 mM IPTG at 37 ºC for 4 h in Rosetta-gami 2 (DE3), and maximum expression for β-1,3-glucanase (89.23 ng/ml) was observed in Rosetta-gami 2 (DE3) with 0.6 mM IPTG at 37 ºC for 6 h. The recombinant endochitinase and β-1,3-glucanase proteins were purified from E. coli using Ni-NTA affinity columns. The purified recombinant proteins were verified by SDS-PAGE, Western blotting and ELISA. The verified recombinant proteins were then tested for antifungal activity by confronting with Rhizoctonia solani casual organism of rice sheath blight and Phytophothora parasitica casual organism of citrus foot rot. The inhibition ratio of R. solani growth by endochitinase protein was 30±2.08 mm and by β-1,3-glucanase was 22.6±0.44 mm. This inhibition was higher than clotrimazole fungicide (21.85±0.47 mm). The recombinant endochitinase did not inhibit growth of P. parasitica whereas, β-1,3-glucanase revealed inhibition ratio of 32.6±0.47 mm against the pathogen. The hydrolytic action of the recombinant endochitinase and β-1,3-glucanase proteins on the cell morphology of R. solani was observed through scanning electron microscopy revealing breakage and pores on hyphal cell walls of R. solani whereas, β-1,3-glucanase confronted hyphae were prone to desiccation and have wrinkles and globular structures on hyphal walls eventually leading to hyphal lysis.ThesisItem Open Access Whole genome de novo assembly of Vigna mungo and Vigna radiata and in silico comparative analysis for marker development(Punjab Agricultural University, Ludhiana, 2018) Thakur, Shivani; Satinder KaurUrdbean and mungbean provides nutritionally balanced food for vegetarian population of South and Southeast Asian countries. But, very less genomic studies are reported in these crops. In this study, we have sequenced (Illumina) and assembled whole genome sequences of V. mungo and V. radiata, covering 559.494 Mb and 588.520 Mb of genome respectively. Gene prediction, functional annotation of predicted genes, identification of (protein families, resistance genes, non-coding RNA genes, transmembrane helix, signal peptide), mapman classification of genes and generation of pseudomolecule was done. To increase genomic resources in these crops, in silico polymorphic marker (SSRs and SNPs) was developed from V. mungo and V. radiata genome sequences . To prove the usefulness of these predicted polymorphisms, validation of these newly designed molecular markers was carried on different Vigna genotypes viz. Vigna radiata, Vigna mungo, Vigna umbellata, Vigna mungo var sylvestris and Vigna radiata var sublobata. Among amplified primers, 91 SSRs and 92 KASPs were polymorphic, representing 85% and 93% of the polymorphism. The performance of these markers was evaluated using various statistical parameters defining the usefulness and robustness of primers. This includes effective multiplex ratio (EMR), resolving power (RP), PIC value and Marker index (MI).Thus, development and validation of these newly designed markers has increased genomic resources in V. mungo and V. radiata. This will benefit genetic mapping, assessment of genetic diversity, marker-assisted selection and translational genomics studies in both these crops. Also, these markers will opens the door to genomic research in other Vigna species.ThesisItem Restricted Genetic Mapping and Transfer of Brown Plant Hopper Resistance Gene(s) from Oryza nivara Sharma et Shastry to Cultivated Rice(Punjab Agricultural University, Ludhiana, 2017) Kishor Kumar; Kumari NeelamBrown planthopper (BPH, Nilaparvata lugen Stål) is one of the most destructive insect of rice (Oryza sativa L.) causing significant yield losses annually. Here, we report high resolution mapping of a novel genetic locus, designated as Bph33 on long arm of rice chromosome 4 using an interspecific F2 population derived from a cross between susceptible indica cultivar PR122 and BPH resistant wild species, O. nivara acc. IRGC104646. Inheritance study was performed using F2, F2:3 and F5 populations revealed presence of single dominant gene. A 50K SNP chip (OsSNPnks) was used to construct high density linkage map and QTL mapping. The resistance locus was mapped between two SNP markers, AX-95952039 and AX-95921548 at LOD score of 28.8 contributing 68.3% of total phenotypic variance. The Bph33 locus spanned a physical distance of 91 Kb in Nipponbare reference genome- IRGSP-1.0 pseudomolecule containing eleven candidate genes, out of which one belong to Leucin Rich Repeat (LRR) family protein. In addition to that, two SSR markers, RM16994 and RM17007 co-segregated with the BPH resistance locus. These SSR markers could be used efficiently for markers assisted transfer of this locus into elite rice cultivars.ThesisItem Restricted Fine mapping and identification of candidate genes for stripe and leaf rust resistance transferred from Aegilops umbellulata to bread wheat (Triticum aestivum)(Punjab Agricultural University, Ludhiana, 2017) Bansal, Mitaly; Chhuneja, ParveenLeaf rust and stripe rust are the most damaging diseases of wheat worldwide. Due to domestication and breeding, the narrow genetic base of modern cultivars makes it difficult to combat the changing environmental conditions as well as the emerging new pathogens. To broaden its genetic base, hybridization with wild relatives, landraces and early domesticates is widely used as they harbor rich genetic diversity. In the present investigation, Lr76 and Yr70 transferred from Ae. umbellulata to bread wheat were fine mapped using next generation sequencing technologies. BC-RIL and F2 population derived from the cross of introgression line pau 16057 (syn. T393-4, IC0616573, INGR 15047) with WL711 segregated for a single gene, each for leaf rust and stripe rust resistance. New SSR and STS markers were designed from the IWGSC survey sequence of chromosome 5D to fine map these genes. Lr57-Yr40_CAPS and 5DS-219 were found to be closely linked to the genes, spanning a region of 5.1cM. To further narrow down the region, SNP based markers were designed from flow sorted chromosome 5D sequence of pau 16057 and WL711. SNPs were identified between the two chromosome sequences and validated through KASP technology. Integrating POPSEQ based ordering of wheat contigs information with SNP discovery in pau 16057 and WL711 sequence, the introgressed segment was found to be locating from 0 to 11.94 cM bin. KASP228 and KASP225 are found to be closely linked to Lr76 and Yr70 and can be used in breeding programmes. We have also established that Lr76 and Yr70 are different from previously mapped Lr57/Yr40 genes in the same region on the basis of their location in 11.94 cM and 4.37 cM POPSEQ bin, respectively. In the present investigation, we have used RenSeq which is based upon the knocking down of the major gene and identification of mutations in the NB-LRR encoding genes in all the mutants of the investigated gene. We have identified two NB-LRR encoding contigs in two mutants of Lr76 which needs further validation. In the present investigation, high end sequencing technology like RenSeq and chromosome sorting and sequencing have been used which made it possible to characterize the introgressed alien segment linked to the rust resistance genes.ThesisItem Open Access Fine Mapping and identification of candidate gene(s) for leaf rust and stripe rust resistance introgressed in Triticum aestivum L. from Aegilops peregrina(Punjab Agricultural University, Ludhiana, 2017) Narang, Deepika; Chhuneja, ParveenAe. peregrina acc. pau 3519, a non-progenitor species (UUSS) maintains high level of resistance to leaf and stripe rust. Two sets of homozygous WL711-Ae. peregrina introgression lines (ILs) viz. IL pau16061 harboring LrAp and IL pau16058 harboring LrP–YrP were crossed with wheat cv. WL711 to generate mapping populations. Inheritance studies, fine mapping of rust resistance genes with SNP/STS markers and identification of candidate genes through Resistance Gene Enrichment Sequencing (RenSeq) were undertaken in the present investigation. F2.3 population of WL711/ IL pau16058 segregated into 260 resistant: 517 segregating: 226 susceptible (χ21:2:1=3.26) against leaf rust and 257 resistant: 520 segregating: 226 susceptible (χ21:2:1=3.281) against stripe rust races indicating transfer of two dominant co-segregating rust resistance genes. In the initial analysis, LrP–YrP genes were mapped on 5DS showing linkage with XLr57/Yr40–CAPS16, Xbarc130 and XTa5DS_44573. High resolution genetic map spanning 4.28cM including eight SNP markers, one SSR and two STS markers was generated. Marker loci BS00163889 flanked distally (co-segregating) and gsnp_5ms_4641 proximally (seven recombinants) to LrP–YrP. SNP markers designed from nucleotide binding and leucine rich repeats (NLRs) depicting independent variations in mutants were placed away from LrP–YrP. Inheritance studies on F6 RILs of WL711/IL pau16061 indicated transfer of a single major dominant gene conditioning seedling resistance. In initial analysis, GISH on IL pau16061 established that LrAp has been introgressed from the Sp genome of Ae. peregrina to the long arm of a pair of homoeologous chromosomes of wheat. Bulked Segregant Analysis (BSA) placed the alien segment onto chromosome 2BL as indicated by linkage of LrAp with markers Xncw-Lr58-1 and Xcfd50. For fine mapping of LrAp, five co-dominant SNP markers and one dominant marker based on presence/absence variations were designed directly from NLRs. These markers positioned the LrAp gene distally on the alien segment and indicated homology with the 6BL chromosome of wheat. The marker Ren_1191 showed co-segregation with LrAp. Linkage of both 2BL and 6BL specific markers with LrAp indicated translocation of a 6Sp alien segment onto the long arm of wheat chromosome 2B. The identification of co–segregating markers for LrAp and LrP–YrP in the present study will facilitate marker–assisted pyramiding with other genes.
